The mapping and sequencing of human genome is a world-wide effort with many anticipated health benefits. A cornerstone of the Human Genome Project (HGP) is the use of yeast artificial chromosome (YAC) vectors for the cloning of large chromosome fragments. It is essential for the HGP that human DNA within YACs be stable within yeast. A major source of artefacts as we have shown arises from the repetitive nature of tie DNA. These are potential substrates for co-cloning events and intra-YAC mitotic recombination since recombination between diverged DNAs has been demonstrated in yeast. Some YACs exhibit considerable internal recombinational repair. We have suggested that the extent of repeats, even if diverged, may be indicated by the sensitivity of YACs to ionizing radiation induced loss and that the survival of YACs is dictated by the amount of recombinational repair between the diverged DNAs (currently under study). Small repeats that surround large inverted repeats (such as Alu's) may be sites of excision, as we have shown for Tn5 DNA in yeast. Problems in replication may also contribute significantly to YAC instability. The only yeast origins of replication are present at one or both telomeres. These are insufficient for replication of large human DNA containing YACs. Presumably replication relies on consensus origins in human DNAs. These may be insufficient or of limited function. As we have shown, altered replication is known to enhance recombination between large direct repeats in chromosomes and, it stimulates as much as 100-fold excision involving small repeats in Tn5. The goals of the project are: 1) improvement of cloning methods and recipient strains, 2) improvement of YAC stability utilizing chromosome stability/transmission and recombination mutants and gene products, and 3) development of in vivo fragmentation systems for the precise physical mapping of sequences of interest within YACs. We are also merging YAC cloning systems with mammalian viral cloning systems for the easy transfer of large fragments to human cells.